The World Health Organization estimates that 2 million people die every year from tuberculosis and that 30% of the world's population is infected. Multi-drug resistant organisms are widespread and extensively drug resistant organisms are emerging. New approaches are required to combat these virulent and drug resistant organisms (MDR/XDR TB). The function of Rv3409c is unknown. It is distantly related to bacterial cholesterol oxidases and was annotated as a cholesterol oxidase during genome sequencing. Experiments with purified protein and transposon mutants revealed that Rv3409c is not a cholesterol oxidase. The mutant has a colony morphology phenotype and lipid profiling has identified lipids present in the mutant that are not present in wild-type mycobacteria. The proposed experiments are aimed at characterizing Rv3409c. The hypothesis is that Rv3409c is a glycolipid oxidase and that this activity represents a new target for anti-MDR-TB pharmaceutical development. The aim of this work is 1) to elucidate the structure of lipids unique or missing in the Rv3409c mutant and 2) to determine the kinetic activity and substrate specificity of the Rv3409c enzyme. PUBLIC HEALTH RELEVANCE: The proposed research fits within the targeted research needs of NIAID that seeks the development of new chemotherapeutic agents against MDR/XDR TB. These experiments will provide a basis for discovering the function of this enzyme that is not present in humans and guide development of new antibiotics that would be effective against all forms of TB including MDR/XDR TB.